Description (Adapted from Application): The presence of bacterial indicators of biological water quality is commonly determined by the use of culturing techniques. However, indicator bacteria can not always grow on artificial media due to injury or stress imposed by prevailing environmental conditions. Consequently, culturing methods often underestimate the in situ densities of indicator bacteria. It is therefore necessary to use methods that do not exclusively rely on microbial growth in artificial media to accurately determine microbial densities. The proposed work deals with the development of nucleic acid based methods to determine the presence and survival of enteric bacteria in tropical recreational waters. One method will use fluorescently labeled rRNA targeting probes in whole cell hybridization studies. Initial studies will evaluate the sensitivity of this assay using eubacterial rDNA probes against bacterial indicators of fecal contamination (e.g., Escherichia coli, Enterococcus faecalis). A specific effort will be focused on developing a resuscitation approach to increase the sensitivity of the probing method. The rDNA probing technology will then be used to determine survival rates of E. coli in recreational waters using membrane diffusion chambers. An alternate molecular approach to monitor bacterial cells will consist of the development of an in situ PCR technique. This method will use rRNA and mRNA transcripts as targets and therefore it will be specific for active cells. A epifluorescence microscope linked to a charge coupled device and image analysis software will be used to further increase the sensitivity of both whole cell hybridization and in situ PCR assays and to decrease errors due to cell autofluorescence and background noise (i.e., fluorescing debris). This approach will also provide a quantitative estimation of the different levels of activity of enteric microorganisms, including pathogenic bacteria, in complex environmental samples. This information is essential to better estimate the risks associated with the presence of pathogens in tropical recreational waters.